Genotyping of canine MHC gene DLA-88 by next-generation sequencing reveals high frequencies of new allele discovery and gene duplication.

Dogs have served as one of the most reliable preclinical models for a variety of diseases and treatments, including stem/progenitor cell transplantation. At the genetic epicenter of dog transplantation models, polymorphic major histocompatibility complex (MHC) genes are most impactful on transplantation success. Among the canine class I and class II genes, DLA-88 has been best studied in transplantation matching and outcomes, with 129 DLA-88 alleles identified. In this study we developed and tested a next generation (NGS) sequencing protocol for rapid identification of DLA-88 genotypes in dogs and compared the workflow and data generated with an established DLA-88 Sanger sequencing protocol that has been in common prior use for clinical studies. By testing the NGS protocol on a random population of 382 dogs, it was possible to demonstrate superior efficacy based on laboratory execution and overall cost. In addition, NGS proved far more effective at discovering new alleles and detecting multiple alleles associated with gene duplication. A total of 51 new DLA-88 alleles are reported here. This rate of new allele discovery indicates that a large pool of yet un-discovered DLA-88 alleles exists in the domestic dog population. In addition, more than 46% of dogs carried three or more copies of DLA-88, further emphasizing the need for more sensitive and cost-effective DLA typing methodology for the dog clinical model.

The canine gut microbiome is associated with higher risk of gastric dilatation-volvulus and high risk genetic variants of the immune system.

BACKGROUND: Large and giant dog breeds have a high risk for gastric dilatation-volvulus (GDV) which is an acute, life-threatening condition. Previous work by our group identified a strong risk of GDV linked to specific alleles in innate and adaptive immune genes. We hypothesize that variation in the genes of the immune system act through modulation of the gut microbiome, or through autoimmune mechanisms, or both, to predispose dogs to this condition. Here, we investigate whether differences in the canine fecal microbiome are associated with GDV and are linked to previously identified risk alleles. METHODOLOGY/PRINCIPLE FINDINGS: Fecal samples from healthy Great Danes (n = 38), and dogs with at least one occurrence of GDV (n = 37) were collected and analyzed by paired-end sequencing of the 16S rRNA gene. Dietary intake and temperament were estimated from a study-specific dietary and temperament questionnaire. Dogs with GDV had significantly more diverse fecal microbiomes than healthy control dogs. Alpha diversity was significantly increased in dogs with GDV, as well as dogs with at least one risk allele for DRB1 and TRL5. We found no significant association of dietary intake and GDV. Dogs with GDV showed a significant expansion of the rare lineage Actinobacteria (p = 0.004), as well as a significantly greater abundance of Firmicutes (p = 0.004) and a significantly lower abundance of Bacteroidetes (p<0.004). There was a significant difference in the abundance of 10 genera but after correction for multiple comparisons, none were significant. Bacterial phyla were significantly different between controls and dogs with GDV and at least one risk allele for DRB1 and TRL5. Actinobacteria were significantly higher in dogs with GDV and with one risk allele for DRB1 and TLR5 but not DLA88 genes. Furthermore, Collinsella was significantly increased in dogs with at least one risk allele for DRB1 and TLR5. Logistic regression showed that a model which included Actinobacteria, at least one risk allele,and temperament, explained 29% of the variation in risk of GDV in Great Danes. CONCLUSIONS: The microbiome in GDV was altered by an expansion of a minor lineage and was associated with specific alleles of both innate and adaptive immunity genes. These associations are consistent with our hypothesis that immune genes may play a role in predisposition to GDV by altering the gut microbiome. Further research will be required to directly test the causal relationships of immune genes, the gut microbiome and GDV.

Associations between gastric dilatation-volvulus in Great Danes and specific alleles of the canine immune-system genes DLA88, DRB1, and TLR5.

OBJECTIVE To determine whether specific alleles of candidate genes of the major histocompatibility complex (MHC) and innate immune system were associated with gastric dilatation-volvulus (GDV) in Great Danes. ANIMALS 42 healthy Great Danes (control group) and 39 Great Danes with ≥ 1 GDV episode. PROCEDURES Variable regions of the 2 most polymorphic MHC genes (DLA88 and DRB1) were amplified and sequenced from the dogs in each group. Similarly, regions of 3 genes associated with the innate immune system (TLR5, NOD2, and ATG16L1), which have been linked to inflammatory bowel disease, were amplified and sequenced. Alleles were evaluated for associations with GDV, controlling for age and dog family. RESULTS Specific alleles of genes DLA88, DRB1, and TLR5 were significantly associated with GDV. One allele of each gene had an OR > 2 in the unadjusted univariate analyses and retained a hazard ratio > 2 after controlling for temperament, age, and familial association in the multivariate analysis. CONCLUSIONS AND CLINICAL RELEVANCE The 3 GDV-associated alleles identified in this study may serve as diagnostic markers for identification of Great Danes at risk for GDV. Additional research is needed to determine whether other dog breeds have the same genetic associations. These findings also provided a new target for research into the etiology of, and potential treatments for, GDV in dogs.

Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line.

The dog is recognized as a highly predictive model for preclinical research. Its size, life span, physiology, and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model has not been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for preclinical models of genetic diseases. Specifically, we i) established markers for identification and tracking canine spermatogonial cells; ii) established methods for enrichment and genetic manipulation of these cells; iii) described their behavior in culture; and iv) demonstrated engraftment of genetically manipulated SSC and production of transgenic sperm. These findings help to set the stage for generation of transgenic canine models via SSC transplantation.

Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

A preclinical model of double- versus single-unit unrelated cord blood transplantation.

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained

Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis.

Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix. Using artificial mixtures of retrovirally marked, single-cell-derived clones, we demonstrate that LAM-PCR fails to detect 30-40% of the clones, even after exhaustive analysis. Furthermore, the relative abundance of specific clones within a mix is not accurately represented, deviating by as much as 60-fold from their true abundance. We describe an optimized, multiarm, high-throughput modification of LAM-PCR that improves the global detection capacity to greater than 90% with exhaustive sampling, facilitates accurate estimates of the total pool size from smaller samplings, and provides a rapid, cost-effective approach to the generation of large insertion-site data bases required for evaluation of vector integration preferences. The inability to estimate the abundance of individual clones within mixtures remains a serious limitation. Thus, although LAM-PCR is a powerful tool for identification of integration sites and for estimations of clonal complexity, it fails to provide the semiquantitative information necessary for direct, reliable tracking of individual clones in a chimeric background.

Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning.

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells.

The human osteosarcoma-derived cell line, SAOS-2, exhibits many of the phenotypic characteristics of osteoblasts including the deposition of types I and V collagens in an extracellular matrix. Lesser amounts of collagen XI chains were also detected. The cell layer collagen contains hydroxylysyl pyridinoline cross-links but without the accompanying lysyl pyridinoline typical of human bone collagen. This indicates that the lysine residues at the two helical cross-linking loci are fully hydroxylated. The isoform of lysyl hydroxylase, LH1, known to be required for full hydroxylation at these sites, was shown to be highly expressed by SAOS-2 cells. Our findings provide insight on the mechanism of post-translational overmodification of lysine residues in collagen made by osteosarcoma tumors, and may be relevant for understanding a similar overmodification observed in osteoporotic bone.

Growth factor receptors as regulators of hematopoiesis.

Nearly 15 years have elapsed since the US Food and Drug Administration last approved a major new hematopoietic cytokine. Promiscuous binding to multiple receptors, or to receptors expressed by multiple tissues, reduces growth factor specificity and promotes side effects. Here we show that hematopoiesis can be differentially regulated using receptors rather than ligands. Conditional derivatives of both fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) induced a sustained expansion of mouse marrow cells ex vivo, and erythroid cells in vivo. Only F36VFGFR1 could support the ex vivo expansion of short-term repopulating hematopoietic stem cells (HSCs), the ex vivo survival of long-term repopulating HSCs, and the prolonged in vivo expansion of granulocytes, monocytes, and platelets. Only F36VMpl induced a response sufficiently rapid to accelerate recovery from radiation-induced anemia. These results establish receptors as a new class of hematopoietic regulators possessing activities unobtainable with growth factors.

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal.

Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate. Only differentiating erythroid precursors and multipotential and erythroid progenitors (colony-forming unit [CFU]-mix and burst forming unitserythroid [BFUe]) responded; however, the nature of the response differed markedly between bone marrow and spleen. In the marrow, F36VMpl induced a 12- to 17-fold expansion of differentiated erythroid precursors and a loss of CFU-mix and BFUe. In the spleen, F36VMpl induced a marked rise in BFUe and CFU-mix and, relative to marrow, a much less prominent rise in more mature red cells. Clonal analysis was most consistent with the interpretation that the spleen and bone marrow differentially regulate the response of human progenitors to a mitotic signal, possibly influencing progenitor expansion versus differentiation. These findings establish CIDs as in vivo growth factors for human hematopoietic cells.

Overexpression of glutathione-S-transferase, MGSTII, confers resistance to busulfan and melphalan.

A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy. The only known route of clearance of busulfan is by GST-mediated conjugation. Using a fibroblast cell line as a model, we have tested the effects of overexpression of three human GST (GSTA1, GSTP1, and MGSTII) on cell survival under a busulfan or melphalan challenge. In two separate assay formats using chronic exposure to busulfan, MGSTII conferred a reproducible twofold selective advantage. GSTA1 and GSTP1 had no effect on busulfan resistance, and melphalan resistance was not affected by expression of any of the GSTs in these assays. In an acute (24-hour) melphalan exposure assay, MGSTII conferred about a twofold selective advantage. Busulfan was not toxic in this assay. RTPCR analysis of human bone marrow CD34+ cells showed that MGSTII is not highly expressed in this stem/early progenitor population. These data indicate that MGSTII may be a useful selective agent in hematopoietic gene therapy.

Radiation dose determines the degree of myeloid engraftment after nonmyeloablative stem cell transplantation.

A multivariate analysis of 121 dogs conditioned with 200, 100, or 50 cGy of total body irradiation (TBI) followed by hematopoietic stem cell transplantation from matched littermates showed that TBI dose was the only factor examined that was statistically significantly associated with the percentage of donor myeloid engraftment in stable long-term chimeras ( P = .008). To understand the direct effects of low-dose irradiation on hematopoietic stem/progenitor cells, nonirradiated and irradiated human CD34 + cells were evaluated for competitive repopulating ability in nonobese diabetic/severe combined immunodeficiency beta2m -/- mice. As expected, the results showed a radiation dose-dependent loss of competitive repopulating ability. Flow cytometric analysis indicated that, within a viable cell gate, there was reduced expression of P-selectin glycoprotein ligand-1 and L selectin on irradiated compared with nonirradiated CD34 + cells; this suggests that irradiated stem/progenitor cells may be compromised in their ability to home to or interact with the marrow microenvironment. However, the CD34 + /P-selectin glycoprotein ligand-1 dim cells also showed activation of caspase-3, indicating that they were destined to die. These results suggest that the TBI dose determines the degree of myeloid engraftment by compromising the resident stem/progenitor cell compartment.

Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.

The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.

A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side "population" (SP) of bone marrow. This SP from bone marrow, as well as other tissues, is reported to contain immature stem cells with considerable plasticity. Some cell lines also efflux Hoechst and generate SP profiles. Reverse transcription-polymerase chain reaction (RT-PCR) and efflux inhibition studies with the lung carcinoma cell line, A549, implicated the ABCG2 transporter as a Hoechst efflux pump. Furthermore, it is shown that transient expression of ABCG2 generates a robust SP phenotype in human embryonic kidney (HEK293) cells. The results allow the conclusion that ABCG2 is a potent Hoechst efflux pump. Semiquantitative RT-PCR was used to characterize the developmental pattern of expression of ABCG2 in hematopoiesis. It is expressed at relatively high levels in putative hematopoietic stem cells (isolated as SP, 34+/38- or 34+/KDR+ populations) and drops sharply in committed progenitors (34+/38+, 34+/33+, or 34+/10+). Expression remains low in most maturing populations, but rises again in natural killer cells and erythroblasts. Comparison of messenger RNA (mRNA) levels for the 3 major multidrug-resistant efflux pumps, MDR1, MRP1, and ABCG2, in bone marrow SP cells reveals that ABCG2 is the predominant form in these cells. These data suggest that ABCG2 contributes significantly to the generation of the SP phenotype in hematopoietic stem cells. Furthermore, the sharp down-regulation of ABCG2 at the stage of lineage commitment suggests that this gene may play an important role in the unique physiology of the pluripotent stem cell.